The designed enzymatic synthesis of oligosaccharide-containing molecules has recently gained prominence in the art as greater numbers of glycosyltransferase enzymes have become available to skilled workers. See, for instance, U.S. Pat. No. 5,180,674 and allowed U.S. patent application Ser. No. 07/670,701, filed Mar. 18, 1991.
Indeed, U.S. Pat. No. 4,569,909 to Seno et al. teaches the use of uridine diphosphate-N-acetylglucosamine 4-epimerase to epimerize UDP-GlcNAc into an equilibrium mixture of UDP-GlcNAc and UDP-GalNAc. That mixture, after boiling to stop enzymic activity and centrifugation to remove the denatured enzyme, provided a “rough” preparation of UDP-GalNAc that was used with an α-N-acetylgalactosaminyl transferase referred to as “A-transferase” to convert Type O red blood cells into Type A red blood cells.
Seno et al. began each of their reactions with UDP-GlcNAc, a compound that is relatively difficult to prepare and store in large quantity. Seno et al. also had no concept of a regeneration step in which UDP-GalNAc or any other sugar-linked nucleotide is recycled.
Oligosaccharide synthesis based on sugar nucleotide-dependent glycosyltransferases proceeds regio- and stereoselectively under mild reaction conditions without multiple protection and deprotection step. For review in the field, see Toone, et al., Tetrahedron, 45:5365 (1989); David et al., Adv. Carbohydr. Chem. Biochem., 49:175 (1991); Drueckhammer et al., Synthesis, 7:499 (1991) and Ichikawa et al., Anal. Biochem., 202:215 (1992). Glycosyltransferases, however, are difficult to obtain (β-1,4-galactosyltransferase is the only one commercially readily available), and the enzymatic synthesis requires sugar nucleotide regeneration for large-scale processes. Wong et al., J. Org. Chem., 47:5416 (1982); Ichikawa et al., J. Am. Chem. Soc., 113:4698 (1991); Ichikawa et al., J. Am. Chem. Soc., 113:6300 (1991); Wong et al., J. Org. Chem., 57:4343 (1992); Ichikawa et al., J. Am. Chem. Soc., 114:9283 (1992).
More than 50 glycosyltransferase genes have been cloned and sequenced from bacteria, yeast and mammalian cells, and documented in the Genebank (IntelliGenetics, Inc.). The availability of these sequences provides researches an opportunity to overexpress glycosyltransferases in large quantities and use them for oligosaccharide synthesis. Of the eight sugar nucleotides commonly used as donor substrates for mammalian glycosyltransferases, five; i.e. UDP-Glc, UDP-Gal, GDP-Fuc, CMP-NeuAc and UDP-Glucuronic acid, have the regeneration system available for large-scale processes. For a review, see Wong et al., Pure & Appl. Chem., 64:1197-1202 (1992).
The enzymes required for the regeneration of GDP-Man, UDP-GlcNAc and UDP-GalNAc have been reported, [Heidlas et al., Acc. Chem. Res., 25:307-314 (1992); Wong et al., Pure & Appl. Chem., 64:1197-1202 (1992)] although regeneration of these sugar nucleotides has not been demonstrated. As part of efforts to develop glycosyltransferase-based enzymatic procedures for the synthesis of complex oligosaccharides and glycopeptides are disclosed hereinafter, as are the overproduction and specificity study of the soluble catalytic domain of an α1,2-mannosyltransferase (ManT), [Lewis et al., Glycobiology, 2:77 (1992)]. The application of this enzyme coupled with regeneration of guanosine 5′-diphosphomannose (GDP-Man) to the synthesis of mannose-containing oligosaccharides and glycopeptides are also disclosed hereinafter, and by the present inventor and colleagues in Wong et al., Pure & Appl. Chem., 65(4):803-809 (1993) and Wang et al., J. Org. Chem., 58:3985-3990 (1993).